A 32 kDa Mycoplasma pneumoniae protein has been shown to bind specifically to a 100 kDa asialoglycoprotein receptor on the MRC-5 human lung fibroblasts and facilitate attachment. Rabbit anti-32 kDa antiserum is capable of inhibiting attachment of 14C-labelled M. pneumoniae to MRC-5 fibroblasts by 50%. This protein was shown to bind specifically and avidly to a dextran sulfate affinity column, suggesting it represents an M. pneumoniae adhesin that attaches to the sulfated glycoconjugate class of receptors for this pathogen. Surface proteolysis experiments demonstrated the surfaced exposed nature of this protein, while Triton X-114 phase partitioning of the 32 kDa protein into the hydrophobic phase suggests it is an integral membrane protein. It has an isoelectric point of between 5.8 and 5.9. Immunoblots of the proteins with convalescent M. pneumoniae patient sera show that this protein is antigenic and stimulates an immune response which varies with the individual patient. The gene encoding the 32 kDa protein was cloned into the expression vector lgt11. Expressing clones were detected immunologically by reaction with rabbit anti-32 kDa serum.